Functional affinity (avidity) is a measure of the overall binding strength of an antigen with many antigenic determinants. Polymerization of antigen-binding partners greatly increases their availability (or valency) for binding to a group of specific identical ligands in very close proximity to a target cell, resulting in greater target binding strength, slow dissociation rate and cross-linking effect which can prolong modulation of the ligands and facilitate biological potency.
A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, linked by a short linker peptide. A major disadvantage of scFv, in comparison with the bivalent immunoglobulin G (IgG) counterpart, is the monovalency of the product, which precludes an increased avidity due to polyvalent binding. Several strategies have been developed for the multimerization of scFv in order to increase avidity.
Recombinant production of a trivalent single-chain antibody fragment (scFv) fusion protein by using a trimerization domain, including a C-propeptide of procollagens, a coiled-coil neck domain of collectin family proteins, a C-terminal portion of FasL and a bacteriophage T4 fibritin foldon domain (Hoppe, H. J., P. N. Barlow, et al. (1994). “A parallel three stranded alpha-helical bundle at the nucleation site of collagen triple-helix formation.” FEBS Lett 344(2-3): 191-195; Frank, Kammerer et al. 2001 “Stabilization of short collagen-like triple helices by protein engineering.” J Mol Biol 308(5): 1081-1089; Holler, N., A. Tardivel, et al. (2003). “Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.” Mol Cell Biol 23(4): 1428-1440.) has been reported. A short alpha-helical collagen-like peptide capable of self-trimerization and propagation of the heterologous fusion proteins from either the C- or N-terminal direction has also been reported in EP1798240B1. The heterologous fusion domains used in EP1798240B1 were presented in scFv antibody fragments. However, there are disadvantages of scFv in multivalent formats for therapeutic applications.
Unlike immunoglobulin G (IgG) molecules which can be easily purified by affinity chromatographies on protein A or G-conjugated resins through binding to the Fc fragment of IgG, resulting in more than 98% in homogeneity of the product at the first step of purification scheme, purification of the multimeric scFv fusions for therapeutic applications is challenging work since no commercial affinity columns are available. Multivalent scFvs have significantly different stabilities depending on the specific variable domains from which they are constructed (Jung, S., A. Honegger, et al. (1999). “Selection for improved protein stability by phage display.” J Mol Biol 294(1): 163-180; Worn, A. and A. Pluckthun (2001). “Stability engineering of antibody single-chain Fv fragments.” J. Mol Biol 305(5): 989-1010). It has been reported that multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange, leading to heterogeneous antibody variants (Wu, A. M., G. J. Tan, et al. (2001). “Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange.” Protein Eng 14(12): 1025-1033).
CFY196 is composed of an Fab fragment of a humanized version of mAb 1A616 fused with a linker derived from human immunoglobulin D hinge and a tetramerization domain derived from the coiled-coil sequence of human transcription factor ATFα (Charles, Luo et al. 2003). However, ATFα is not a plasma-derived protein, which may associate with the risk of an immune response that could severely limit potential therapeutic applications.
U.S. Patent Application Publication U.S. 2008/0176247 demonstrates that an anti-CD3 scFv N-terminal fused to a self-trimerization collagen-like scaffold comprising GPP triplets is capable of forming a trimeric antibody fragment made up of three single-chain peptides. However, downstream purification of these trimeric scFv versions of collagen-like scaffold fusions was cumbersome since there is no available affinity resin to purify it efficiently. Additionally, the low protein expression level and the thermal instability of said trimeric scFv versions were not qualified to be used for biotherapeutics. Therefore, there is a need to design a new format of trimeric collagen scaffold antibodies.